Zhang_2014_Appl.Environ.Microbiol_80_7348

A novel nonheme chloroperoxidase (RhEst1), with promiscuous esterase activity for enantioselective hydrolysis of ethyl (S)-2,2-dimethylcyclopropanecarboxylate, was identified from a shotgun library of Rhodococcus sp. strain ECU1013. RhEst1 was overexpressed in Escherichia coli BL21(DE3), purified to homogeneity, and functionally characterized. Fingerprinting analysis revealed that RhEst1 prefers para-nitrophenyl (pNP) esters of short-chain acyl groups. pNP esters with a cyclic acyl moiety, especially that with a cyclobutanyl group, were also substrates for RhEst1. The Km values for methyl 2,2-dimethylcyclopropanecarboxylate (DmCpCm) and ethyl 2,2-dimethylcyclopropane carboxylate (DmCpCe) were 0.25 and 0.43 mM, respectively. RhEst1 could serve as an efficient hydrolase for the bioproduction of optically pure (S)-2,2-dimethyl cyclopropane carboxylic acid (DmCpCa), which is an important chiral building block for cilastatin. As much as 0.5 M DmCpCe was enantioselectively hydrolyzed into (S)-DmCpCa, with a molar yield of 47.8% and an enantiomeric excess (ee) of 97.5%, indicating an extremely high enantioselectivity (E = 240) of this novel and unique biocatalyst for green manufacturing of highly valuable chiral chemicals.